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Knockout Mice1
Departments of
*
Oral Biology and
Microbiology, The Immunobiology Vaccine Center, University of Alabama at Birmingham, Medical Center, Birmingham, AL 35294; and
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production with impaired levels of IL-2, IL-4,
IL-5, and IL-10. These mice possessed reduced splenic OVA-specific T
cell proliferative and delayed-type hypersensitivity responses when
compared with nontolerized controls. Further, OVA-specific IgG Ab
responses in serum and the numbers of IgG Ab-forming cells in spleen
were significantly diminished following systemic challenge with OVA in
CFA. When IFN--deficient (IFN--/-) mice of
the same genetic background were given an oral dose of 25 mg of OVA
before systemic immunization, no reduction in OVA-specific IgG Ab
responses in serum and spleen was seen. Furthermore, the serum IgG Ab
responses were restricted to IgG1 and IgG2b subclasses. Interestingly,
although IFN--/- mice displayed a partial diminishment
of T cell proliferative and delayed-type hypersensitivity responses to
OVA, significant responses were still present when compared with the
low responses noted in IFN-+/+ mice. In addition,
OVA-specific T cells from IFN--/- mice produced
Th2-type cytokines (e.g., IL-4), which provided help for systemic
OVA-specific serum IgG1 and IgG2b Ab responses. These findings clearly
indicate a central role for IFN- in the induction and maintenance of
mucosally induced tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of tandem cytokine production by murine CD4+ Th
cell clones has provided direct evidence that Th cells can be divided
into two distinct classes, Th1- and Th2-type cells, on the basis of the
pattern of cytokines secreted (16). It is well established that
CD4+ Th1-type cells secrete IFN-, IL-2, and
lymphotoxin-
for cell-mediated immunity and delayed-type
hypersensitivity (DTH),3
whereas CD4+ Th2-type cells preferentially secrete IL-4,
IL-5, IL-6, IL-10, and IL-13, which provide help for humoral Ab
responses of different isotypes (16, 17, 18). In view of the distinct
characteristics displayed during the immune response (e.g.,
cell-mediated immunity or DTH vs Ab isotype/subclass responses), it
seemed plausible that Th1- and Th2-type cells also play different roles
in mucosally induced tolerance (19). Recent studies have suggested that
Th1-type cells appear to be more susceptible to the induction of
tolerance than Th2-type cells (20), as manifested by their diminished
IgG2a responses and reduced IL-2 and IFN- production (21). Others
have shown that tolerance could be induced in IL-4 knockout mice that
have a dominant Th1 with defective Th2 responses when a high oral dose
of Ag was administered (22); however, the precise mechanisms for
tolerance remain undefined.
In this study, we have adopted an experimental system of IFN-
gene-disrupted (IFN--/-) and normal
(IFN-+/+) mice to elucidate the role of Th1 and Th2
cells in mucosally induced tolerance. When mice of these two types were
given a high oral dose of OVA, clear differences in the level of
tolerance induction of T and B cells were noted. Our results support a
central role for IFN- in the induction of mucosally induced
tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All BALB/c and C57BL/6 mice used in our experiments were
obtained from The Jackson Laboratory (Bar Harbor, ME). Both
IFN--/- on a BALB/c background and
IFN--/- on a C57BL/6 background were used between the
ages of 8 and 12 wk in our experiments. The BALB/c
IFN--/- mice were generously provided by Dr. Timothy
A. Stewart of Genentech (San Francisco, CA; 23 and the C57BL/6
IFN--/- mice were obtained from The Jackson
Laboratory. The IFN--/- mice were genotyped by PCR.
All mice were provided sterile food and water ad libitum and maintained
under pathogen-free conditions in a flexible Trexler isolator (Standard
Safety Equipment Co., Palatine, IL).
Immunization protocols
To induce systemic unresponsiveness to OVA (Fraction V; Sigma Chemical Co., St. Louis, MO), mice were given 25 mg of OVA dissolved in 0.25 ml of PBS by gastric intubation. Control mice received PBS only. On the 7th and 21st days following intubation, mice were challenged by the s.c. route with 100 µg of OVA in 100 µl of CFA (Difco Laboratories, Detroit, MI) to ensure the development of mucosally induced tolerance. DTH responses and serum Ab titers were measured 7 days after the second s.c. immunization. In most studies, the experiment was terminated at this point for analysis of OVA-specific B and T cell responses.
OVA-specific serum Abs by ELISA
Anti-OVA Ab titers in serum samples were determined by ELISA as
described previously (24). Briefly, Falcon Microtest III assay plates
(Becton Dickinson, Oxnard, CA) were coated overnight at 4°C with 1
mg/ml of OVA in PBS. Blocking was done with 200 µl of 1% BSA in PBS
for 1 h at 37°C. Serial dilutions of serum in 1% BSA/PBS were
prepared and 100 µl was added per well in duplicate. Following
incubation at 37°C for 4 h, the horseradish peroxidase-labeled
anti-mouse µ, , and heavy chain-specific Abs (Southern
Biotechnology Associates, Birmingham, AL) were added and incubated
overnight at 4°C. For IgG subclass determinations, biotin-conjugated
mAbs specific for IgG1, IgG2a, IgG2b, and IgG3 (PharMingen, San Diego,
CA) and peroxidase-labeled anti-biotin Ab (Vector Laboratories,
Inc., Burlingame, CA) were employed. Color was developed with 1.1 mM
2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma) in 0.1
M citrate-phosphate buffer (pH 4.2) containing 0.01%
H2O2.
Enumeration of Ab-forming cells by enzyme-linked immunospot (ELISPOT)
An ELISPOT assay was used to detect OVA-specific IgM, IgG, and
IgA Ab-forming cells (AFC) in spleen from immunized mice as described
previously (24, 25). Briefly, 96-well nitrocellulose plates (Millititer
HA; Millipore Corp., Bedford, MA) were coated with OVA (1 mg/ml) in PBS
(100 µl/well) overnight at 4°C. Wells were blocked with PBS
containing 3% BSA. After addition of appropriate dilutions of spleen
cells in RPMI 1640 containing 10% heat-inactivated FCS to individual
wells, the plates were incubated for 4 h at 37°C with 5%
CO2. The AFC were detected with peroxidase-labeled
anti-mouse µ-, -, and -chain-specific Abs (Southern
Biotechnology Associates) and visualized by adding the chromogenic
substrate, 3-amino-9-ethylcarbazole (Moss, Inc., Pasadena, MA). Spots
representing AFC were counted with the aid of a dissecting microscope
(SZH Zoom Stereo Microscope Systems, Olympus, Lake Success,
NY).
In vitro assay for B cell unresponsiveness
To simultaneously perform in vitro and in vivo experiments,
spleen cells were isolated from orally treated
IFN--/- and IFN-+/+ mice following
a single systemic immunization. Splenic mononuclear cells were prepared
by a standard protocol as described previously (24, 25). These
mononuclear cells (5 x 106 cells/ml) were cocultured
with OVA (1 mg/ml) for 5 days in vitro. Nonadherent cells were
harvested and then subjected to the OVA-specific ELISPOT assay
described above.
Measurement of DTH responses
A standard protocol for the measurement of DTH responses was employed in this study (26). Briefly, 10 µg of OVA in 20 µl of PBS were injected into the left ear pinna; the right ear pinna received PBS as a control. Ear swelling was measured 48 h later with a dial thickness gauge (Ozaki Mfg. Co., Ltd., Tokyo, Japan). The DTH response was expressed as the increase of ear swelling after OVA injection following subtraction of swelling in the control site injected with PBS.
OVA-induced splenic T cell proliferation
Splenic T cells were harvested by using a nylon wool column, and a single cell suspension was prepared in complete medium (RPMI 1640; Cellgro Mediatech, Washington, DC) containing 10% heat-inactivated FCS, HEPES buffer (15 mM), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). Splenic T cells (4 x 106 cells/ml) were cultured with 1 mg/ml of OVA in the presence of T cell-depleted, irradiated (3000 rad) splenic feeder cells from naive BALB/c mice in 96-well or 24-well tissue culture plates (Corning Glass Works, Corning, NY) for proliferation assay and cytokine analysis, respectively (27). In some experiments, splenic and Peyer’s patch T cells from mice orally immunized with 25 mg of OVA alone were prepared by a combination of enzymatic digestion (24, 25) and nylon wool column purification, and were then cultured as described above. To measure OVA-specific T cell proliferation, 0.5 µCi of tritiated [3H]thymidine (Amersham Corp., Arlington Heights, IL) was added for the final 18 h of a 4-day culture period. All T cell cultures were incubated at 37°C in a moist atmosphere of 5% CO2 in air.
Cytokine-specific ELISA
Cytokine levels in culture supernatants were determined by ELISA
as previously described (26, 27, 28, 29). Culture supernatants were collected
at 48 h for IFN- and IL-2, and at 96 h for IL-4, IL-5, and
IL-10 production. The immunoplates (Nunc, Inc., Naperville, IL) were
coated with monoclonal anti-IFN- (R4-6A2), anti-IL-2
(JES6-1A12), anti-IL-4 (BVD4-1D11), anti-IL-5 (TRFK-5), or
anti-IL-10 Ab (JES5-2A5) (PharMingen). After blocking with 3% BSA
in PBS, serial twofold dilutions of samples and standards were added to
duplicate wells and incubated overnight at 4°C. The wells were washed
and incubated with the biotinylated monoclonal anti-IFN-
(XMG1.2), anti-IL-2 (JES6-5H4), anti-IL-4 (BVD6-24G2),
anti-IL-5 (TRFK-4), or anti-IL-10 (JES5-16E5) Ab, respectively.
After incubation, peroxidase-labeled anti-biotin Abs (Vector
Laboratories, Burlingame, CA) were added and developed with 1.1 mM
2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma) in 0.1
M citrate-phosphate buffer (pH 4.2) containing 0.01%
H2O2. Standard curves were generated using
mouse rIFN-, rIL-2, rIL-4, rIL-5 (Genzyme, Cambridge, MA), or rIL-10
(PharMingen).
Statistics
The significance of the difference between groups was evaluated by the Mann-Whitney U test for unpaired samples using a Statview II Program designed for Macintosh computers.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-mice
In our initial studies, we determined whether oral administration
of 25 mg of OVA induced B cell unresponsiveness in
IFN-+/+ (BALB/c) mice by assessing OVA-specific
serum Ab titers. Serum from IFN-+/+ mice given OVA by
the oral route possessed significantly lower levels of Ag-specific IgG
Abs than did control mice given oral PBS before s.c. immunization with
OVA in CFA (Fig. 1
). Levels of
OVA-specific IgG subclasses were also assessed and IgG1 was found to be
the most abundant Ag-specific IgG subclass in the serum of mice given
oral PBS following systemic challenge with OVA in CFA but was found in
much lower levels in mice that had been fed OVA. In addition, the
levels of serum OVA-specific IgG2a and IgG2b responses were also
reduced in IFN-+/+ mice orally immunized with OVA, but
the differences were not statistically significant. Based on the
profile of IgG subclass responses, Th2-dependent Ab responses (i.e.,
IgG1) appear to be more susceptible to tolerance induced by a high dose
of OVA.
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gene. As shown in Figure 1
, the magnitude of
the OVA-specific Ab response was generally lower in
IFN--/- than in IFN-+/+ mice. In
contrast to the results for IFN-+/+ mice, the levels of
OVA-specific IgG Abs were essentially unchanged in sera from
IFN--/- mice given oral OVA or PBS. Further, IgG
responses were restricted to IgG1 and IgG2b subclasses in both groups
of IFN--/- mice. Because the possibility existed that
IFN--/- mice might take longer periods for development
of Ag-specific immune responses due to the lack of an appropriate Th
cell environment, a longer interval of OVA-specific Ab responses was
also examined (e.g., up to 4 wk after secondary systemic challenge).
The levels of serum OVA-specific IgG Abs at 2 to 4 wk following
systemic challenge were similar to those seen at the 1- wk interval
(data not shown). These results further demonstrate that
IFN--/- mice orally immunized with a high dose of OVA
do not develop reductions in OVA-specific IgG Ab titers in serum.
To ensure that intact B cell responses were maintained in
IFN--/- mice following oral immunization with a high
dose of OVA, we next investigated the frequency of OVA-specific AFC in
spleen of IFN--/- or IFN-+/+ mice
orally immunized with OVA using an Ag-specific ELISPOT assay. The
numbers of splenic OVA-specific IgG AFC were significantly decreased
(13-fold) in IFN-+/+ mice given oral OVA when compared
with mice that received oral PBS (Table I). In contrast, though the levels of
OVA-specific IgG AFC were slightly lower in the OVA-fed group than
in that treated with PBS (Table I), the decrease was not statistically
significant. An identical pattern of results was obtained when
IFN--/- mice of a different background (e.g., C57BL/6)
were employed. These findings have provided new evidence that
Ag-specific Ab unresponsiveness does not develop following oral
administration of OVA in an environment of IFN- deficiency.
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). OVA-specific IgG AFC were much less
prevalent in spleens from OVA-fed IFN-+/+ mice than in
those from PBS-fed mice. In contrast, the frequency of IgG AFC was not
significantly changed in IFN--/- mice given oral OVA
when compared with the PBS-fed group. Taken together, these results
show that oral administration of a high dose of OVA elicits B cell
unresponsiveness in normal IFN-+/+ mice, but does not
affect OVA-specific B cell responses of IFN--/-
mice.
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-/- mice
To determine the influence of a high dose of oral Ag on T cell
responses in a situation of IFN- deficiency, Ag-specific DTH
responses were assessed after oral administration of OVA to
IFN--/- and IFN-+/+ mice.
Significantly lower Ag-specific DTH responses were noted in
IFN-+/+ mice orally immunized with OVA than in mice
given oral PBS only (Table III). In
contrast, the decreases in Ag-specific DTH responses were modest in
OVA-fed IFN--/- mice when compared with those of
IFN-+/+ mice. The fact that Ag-specific DTH responses
were observed in both groups of IFN--/- mice (Table III) suggests that DTH responses can be induced and maintained even
when Th1-type cells are deficient. Indeed, it was shown that cytokines
other than IFN-, e.g., TNF-, lymphotoxin-, and IL-8, may also
play important roles in Ag-specific DTH responses (30, 31).
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-/- mice fed a high dose of OVA, we examined
OVA-specific T cell proliferative responses in vitro. Splenic T cells
isolated from IFN-+/+ mice orally immunized with OVA
showed significantly lower Ag-specific T cell proliferative responses
than did those from IFN-+/+ mice given PBS orally before
systemic immunization (Table III). Reduced T cell proliferation was
only observed in response to OVA, since Con A-stimulated proliferative
responses were not affected in splenic T cells isolated from either
IFN--/- or IFN-+/+ mice given oral OVA
or PBS (data not shown). On the other hand, although splenic T cells
from IFN--/- mice orally immunized with OVA showed
decreased levels of OVA-specific proliferative responses when compared
with IFN--/- mice given PBS orally, the differences
were not statistically significant (Table III). Taken together, these
findings provide new evidence that oral administration of a high dose
of OVA elicits only partial T cell unresponsiveness in
IFN--/- mice. OVA-induced cytokine responses
Once it was established that oral administration of 25 mg of
OVA resulted in the reduction of OVA-specific T cell proliferative
responses in both IFN-+/+ and
IFN--/- mice, it seemed important to determine the
effects of OVA on cytokine production patterns. Culture supernatants
from OVA-stimulated splenic T cells were examined by ELISA for Th1-
(e.g., IFN- and IL-2) and Th2-type cytokines (e.g., IL-4, IL-5, and
IL-10). Much less IL-4, IL-5, and IL-10 was produced by OVA-stimulated
T cells in culture supernatants from IFN-+/+ mice orally
immunized with OVA than by IFN-+/+ mice given oral PBS
(Fig. 2). These findings are consistent
with the fact that decreased serum IgG1 responses occur in
IFN-+/+ mice given oral OVA. Interestingly, predominant
IFN- production was noted in culture supernatants from
OVA-stimulated splenic T cells of orally tolerized
IFN-+/+ mice. To determine whether the increased IFN-
production was due to a combination of oral OVA followed by OVA and CFA
immunization, IFN-+/+ mice were orally immunized with 25
mg of OVA alone, and T cells from spleen and Peyer’s patches of these
mice were isolated 3 days later. Although T cells isolated from
Peyer’s patches showed Ag-induced cytokine responses including
significant amounts of IFN- synthesis, splenic T cells failed to
produce cytokines. These results suggest that a combination of oral and
systemic immunization elicits a dominant IFN- response by splenic T
cells. These findings provide the first evidence that oral
administration of a high dose of Ag elicits IFN- production by
OVA-stimulated splenic T cells, and that this occurs despite the fact
that T cell proliferative and DTH responses are reduced in orally
tolerized IFN-+/+ mice. Analysis of cytokine synthesis
by Ag-stimulated splenic T cells from IFN--/- mice
orally immunized with OVA revealed low levels of IL-2, IL-4, IL-5, and
IL-10 in culture supernatants when compared with those from mice orally
administered PBS only. However, it is important to point out that
although the levels of IL-4 were reduced in both
IFN--/- and IFN-+/+ mice immunized
orally with OVA, higher IL-4 production was seen in
IFN--/- mice when compared with
IFN-+/+ mice.
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-/- and IFN-+/+ mice.
Interestingly, no significant differences were observed between the
sera from the respective groups of IFN-+/+ and
IFN--/- mice (data not shown). Furthermore, TGF-ß
was not detected in culture supernatants of OVA-stimulated splenic T
cells in vitro, suggesting that TGF-ß may not have an effect on the
induction of systemic unresponsiveness by a high dose of Ag. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+/+ mice but not in identically
treated IFN--/- mice (Fig. 1, Tables I and II).
Although OVA-induced proliferative responses of splenic T cells and DTH
responses were decreased in both IFN-+/+ and
IFN--/- mice following oral administration of OVA,
decreases were more modest in IFN--/- than in
IFN-+/+ mice, which exhibited complete suppression of
proliferative and DTH responses (Table III). When the same experimental
protocol was used in IL-4-/- mice, both T and B cell
unresponsiveness was observed after oral administration of OVA, as
demonstrated by reduced OVA-specific T cell proliferation and DTH
responses and markedly diminished OVA-specific serum IgG production
(our manuscript in preparation). Indeed, it has been shown that Th2
cells are not required for the induction of mucosally induced tolerance
since oral administration of 25 mg of OVA can elicit OVA-specific B and
T cell unresponsiveness in IL-4-/- mice (22). Taken
together, these findings suggest that IFN--producing Th1 cells are
essential for the reduction of systemic B cell responses induced by a
high dose of oral OVA.
Of interest was the finding that IFN- was the only cytokine
demonstrating increased production in culture supernatants of
OVA-stimulated splenic T cells from IFN-+/+ mice given
oral OVA, even though the synthesis of IL-2 and Th2-type cytokines
(e.g., IL-4, IL-5, and IL-10) was significantly reduced (Fig. 2). These
findings strongly suggest that IFN- plays an essential role in the
induction and/or maintenance of systemic tolerance, which is induced at
the B cell level by a high dose of Ag. Thus, it is possible that
production of IFN- by Th1 cells leads to the reduction of IL-4 by
Th2 cells in orally tolerized mice. Subsequently, the lack of IL-4
production may contribute to the low production of Ag-specific IgG
responses, most notably those of the IgG1 subclass. In this regard, it
has been shown that IFN--producing Th1 cells can down-regulate Th2
cell function for IgG1 and IgE responses (16, 18). On the other
hand, IFN--/- mice that were orally administered OVA
showed higher levels of IL-4 (70–80 pg/ml), which favors the
production of IgG1 and IgG2b subclasses, than did
IFN-+/+ mice (5–10 pg/ml) (Fig. 2). Indeed, it has been
shown that Ag-stimulated splenic CD4+ T cells from mice
orally immunized with tetanus toxoid plus cholera toxin induce
approximately 100 pg/ml of IL-4 in vitro, accounting for the subsequent
induction of tetanus toxoid-specific IgG1 Ab responses (26). Among the
several proposed explanations of how orally administered OVA elicits
intact B cell responses in IFN--/- mice, we favor the
one that suggests that IL-4 production by partially tolerized Th2 cells
(as demonstrated by DTH and proliferative responses (Table III)) might
contribute to the maintenance of B cell responses in these
mice.
The role of IFN- in the induction of mucosally induced tolerance is
further supported by recent studies in which spleen cells taken from
mice given OVA by the oral route showed OVA-specific IFN- production
when restimulated with Ag in vitro (32). In addition, a recent study
has demonstrated that repeated oral administration of high doses of OVA
to OVA TCR-transgenic mice results in IFN--dominated immune
responses in the Peyer’s patches (28). Though regulatory mechanisms
for IFN- in the induction and maintenance of mucosally induced
tolerance are not yet known, it is well established that IFN-
contributes to the reduction of IL-4-producing Th2-type cells, which
favor the production of IgG B cell responses (16, 18). Taken together,
the immunologic consequences of systemic B cell tolerance induced by a
high dose of oral Ag could be due to IFN--mediated immune
regulation, with significant suppression of Th2-type cells.
Oral administration of a high dose of OVA to IFN--/-
mice resulted in decreased T cell responses, though this reduced
response was not significant, but B cell responses were intact. A state
of unresponsiveness, or anergy, by immunocompetent cells has been
reported within both T and B cell compartments (33). However,
unresponsiveness of T cells with intact B cell responses has been
reported in humans (34), and a recent study performed in transgenic
mice showed that a small amount of autoantigen induced T cell but not B
cell tolerance (35). The B cell responses can be tolerized but
generally require larger amounts of oral Ag (36, 37). Furthermore, the
kinetics of tolerance induction are fundamentally different in T and B
cells (38). For example, thymocytes become tolerant sooner and remain
so longer than do bone marrow cells after injection of Ag, indicating
that T cells are more easily tolerized and remain anergic longer than B
cells. In this regard, it will be important to investigate the time
course of Ag-specific Ab and T cell responses in both
IFN--/- and IFN-+/+ mice in comparison
to IL-4-/- and IL-4+/+ mice following the
induction of oral tolerance. Our group is currently conducting such
experiments.
Since the level of TGF-ß production had been considered to be the key
factor for mucosally induced tolerance (10, 15, 39), it was important
to examine levels of TGF-ß following oral administration of OVA.
Thus, TGF-ß production in culture supernatants of Ag-stimulated
splenic T cells and total TGF-ß levels in the serum of orally
immunized IFN-+/+ and IFN--/- mice were
assessed by a sensitive ELISA. The levels of TGF-ß were found to be
similar in sera obtained from all groups tested. No detectable levels
of TGF-ß were seen in culture supernatants of OVA-stimulated splenic
T cells isolated from IFN-+/+ or
IFN--/- mice. These findings indicate that mucosally
induced tolerance is not principally mediated by increased synthesis of
TGF-ß in our experimental system. However, others have suggested that
TGF-ß-producing CD4+ T cells (as a Th3 subset) may be
essential for the induction of mucosally induced tolerance with low
doses of Ag (15, 39). This suggestion was further supported by a recent
study that demonstrated the presence of bystander suppression mediated
by TGF-ß after oral administration of low doses of Ag (40). These
findings have indicated that the production of TGF-ß is a necessary
condition for the cytokine-mediated active suppression induced by low
doses of oral Ag. On the other hand, a high dose tolerance was elicited
by clonal anergy or deletion of Ag-specific cells (12, 13). Our present
study has provided an additional possibility that IFN- production
was maintained during the T cell unresponsiveness induced by a high
oral dose of Ag, leading to inhibition of Th2 cells producing IL-4 with
subsequent B cell unresponsiveness, especially for IgG1 subclass
responses.
In summary, our study is the first to reveal that oral administration
of a high dose of Ag induced brisk IFN- production, which may
contribute to suppression of cytokine production (e.g., IL-4) by
Th2-type cells, leading to the induction of B cell unresponsiveness. On
the other hand, the partial maintenance of T cell responses including
IL-4 production as well as intact B cell responses were elicited in
mice with a disrupted IFN- gene, suggesting that IFN- is a key
cytokine for induction and maintenance of mucosally induced
tolerance.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan.
3 Abbreviations used in this paper: DTH, delayed-type hypersensitivity; AFC, Ab-forming cells; IFN--/-, IFN- gene knockout; ELISPOT, enzyme-linked immunospot.
Received for publication January 6, 1997. Accepted for publication October 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene-disrupted mice. J. Exp. Med. 178:2243.This article has been cited by other articles:
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P. M. Cobelens, A. Kavelaars, A. Vroon, M. Ringeling, R. van der Zee, W. van Eden, and C. J. Heijnen The {beta}2-Adrenergic Agonist Salbutamol Potentiates Oral Induction of Tolerance, Suppressing Adjuvant Arthritis and Antigen-Specific Immunity J. Immunol., November 1, 2002; 169(9): 5028 - 5035. [Abstract] [Full Text] [PDF]
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 M-N Kweon, I Takahashi, M Yamamoto, M H Jang, N Suenobu, and H Kiyono Development of antigen induced colitis in SCID mice reconstituted with spleen derived memory type CD4+ CD45RB+ T cells Gut, March 1, 2002; 50(3): 299 - 306. [Abstract] [Full Text] [PDF]
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 A. George-Chandy, K. Eriksson, M. Lebens, I. Nordstrom, E. Schon, and J. Holmgren Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells Infect. Immun., September 1, 2001; 69(9): 5716 - 5725. [Abstract] [Full Text] [PDF]
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D. A. Kahn, D. C. Archer, and C. J. Kelly Absence of Functional Inducible NO Synthase Enhances the Efficacy of Tolerance Induced by High Dose Antigen Feeding J. Immunol., December 1, 2000; 165(11): 6116 - 6122. [Abstract] [Full Text] [PDF]
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M. Yamamoto, P. Rennert, J. R. McGhee, M.-N. Kweon, S. Yamamoto, T. Dohi, S. Otake, H. Bluethmann, K. Fujihashi, and H. Kiyono Alternate Mucosal Immune System: Organized Peyer's Patches Are Not Required for IgA Responses in the Gastrointestinal Tract J. Immunol., May 15, 2000; 164(10): 5184 - 5191. [Abstract] [Full Text] [PDF]
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K. Fujihashi, T. Dohi, M.-N. Kweon, J. R. Mcghee, T. Koga, M. D. Cooper, S. Tonegawa, and H. Kiyono {gamma}{delta}T cells regulate mucosally induced tolerance in a dose-dependent fashion Int. Immunol., December 1, 1999; 11(12): 1907 - 1916. [Abstract] [Full Text] [PDF]
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A. McI. Mowat, M. Steel, A. J. Leishman, and P. Garside Normal Induction of Oral Tolerance in the Absence of a Functional IL-12-Dependent IFN-{gamma} Signaling Pathway J. Immunol., November 1, 1999; 163(9): 4728 - 4736. [Abstract] [Full Text] [PDF]
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 H. Iijima, I. Takahashi, D. Kishi, J.-K. Kim, S. Kawano, M. Hori, and H. Kiyono Alteration of Interleukin 4 Production Results in the Inhibition of T Helper Type 2 Cell-Dominated Inflammatory Bowel Disease in T Cell Receptor {alpha} Chain-Deficient Mice J. Exp. Med., September 6, 1999; 190(5): 607 - 616. [Abstract] [Full Text] [PDF]
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H. N. Shi, M. J. Grusby, and C. Nagler-Anderson Orally Induced Peripheral Nonresponsiveness Is Maintained in the Absence of Functional Th1 or Th2 Cells J. Immunol., May 1, 1999; 162(9): 5143 - 5148. [Abstract] [Full Text] [PDF]
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M.-N. Kweon, K. Fujihashi, Y. Wakatsuki, T. Koga, M. Yamamoto, J. R. McGhee, and H. Kiyono Mucosally Induced Systemic T Cell Unresponsiveness to Ovalbumin Requires CD40 Ligand-CD40 Interactions J. Immunol., February 15, 1999; 162(4): 1904 - 1909. [Abstract] [Full Text] [PDF]
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 A. A. ROSSINI, D. L. GREINER, and J. P. MORDES Induction of Immunologic Tolerance for Transplantation Physiol Rev, January 1, 1999; 79(1): 99 - 141. [Abstract] [Full Text] [PDF]
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) or PBS (•) only, followed by s.c.
immunization with OVA in CFA. The serum was obtained from five mice per
group and tested individually. The results are expressed as the
mean ± SE and show an example from three separate experiments. *,
p < 0.05; **, p < 0.01 vs PBS orally
administered group.
)
or PBS (
) only, followed by s.c. immunization with OVA in CFA.
Splenic T cells (4 x 106 cells/ml) from the immunized
mice were cultured with 1 mg/ml of OVA in the presence of T
cell-depleted and irradiated splenic feeder cells (5 x
106 cells/ml) from naive BALB/c mice. B,
IFN-